BRCA1 germline mutation and glioblastoma development: report of cases Open Access

C A S E R E P O R T Open Access
BRCA1 germline mutation and glioblastoma
development: report of cases
Meriem Boukerroucha
1
, Claire Josse
1,2*
, Karin Segers
3
, Sonia El-Guendi
1
, Pierre Frères
2
, Guy Jerusalem
2
and Vincent Bours
1,3
Abstract
Background: Germline mutations in breast cancer susceptibility gene 1 (BRCA1) increase the risk of breast and
ovarian cancers. However, no association between BRCA1 germline mutation and glioblastoma malignancy has ever
been highlighted.
Here we report two cases of BRCA1 mutated patients who developed a glioblastoma multiform (GBM).
Cases presentation: Two patients diagnosed with triple negative breast cancer (TNBC) were screened for BRCA1
germline mutation. They both carried a pathogenic mutation introducing a premature STOP codon in the exon 11
of the BRCA1 gene. Few years later, both patients developed a glioblastoma and a second breast cancer. In an
attempt to clarify the role played by a mutated BRCA1 allele in the GBM development, we investigated the BRCA1
mRNA and protein expression in breast and glioblastoma tumours for both patients. The promoter methylation
status of this gene was also tested by methylation specific PCR as BRCA1 expression is also known to be lost by this
mechanism in some sporadic breast cancers.
Conclusion: Our data show that BRCA1 expression is maintained in glioblastoma at the protein and the mRNA
levels, suggesting that loss of heterozygosity (LOH) did not occur in these cases. The protein expression is tenfold
higher in the glioblastoma of patient 1 than in her first breast carcinoma, and twice higher in patient 2. In
agreement with the high protein expression level in the GBM, BRCA1 promoter methylation was not observed in
these tumours.
In these two cases, despite of a BRCA1 pathogenic germline mutation, the tumour-suppressor protein expression is
maintained in GBM, suggesting that the BRCA1 mutation is not instrumental for the GBM development.
Keywords: BRCA1, Glioblastoma, Breast cancer
Background
Breast cancer susceptibility gene 1 (BRCA1)isthefirst
tumour suppressor gene identified in familial breast
cancer. Located on chromosome 17 (17q21), this gene
encodes a multifunctional protein involved in several
cellular processes such as DNA repair, chromatin re-
modelling and cell cycle regulation [1]. Several studies
reported that germline mutations in BRCA1 gene in-
crease the risk to develop breast and ovarian cancers
[1,2]. Indeed, women bearing pathogenic germline
BRCA1 mutations have a 45% to 80% risk to develop
breast cancer by age 70, and 36% to 66% for ovarian
cancer [3]. BRCA1 somatic mutations are very rare but
its promoter methylation is reported to occur in about
7% to 30% of breast and ovarian sporadic cancers [4,5].
In glioma, genome-wide association studies have iden-
tified common genetic variations in 7 genes that in-
crease glioma risk (TERT, EGFR, CCDC26, CDKN2A,
CDKN2B, PHLDB1 and RTEL1)butBRCA1 is not
among them and there is no known association be-
tween BRCA1 gene and glioblastoma multiforme
(GBM) [6-8]. Indeed, Elmariah and co-authors reported
the case of a patient mutated for BRCA1 who devel-
oped glioblastoma but they did not investigate BRCA1
mRNA and protein expression in the GBM [9]. Piccirilli
and co-authors have also studied 11 cases of GBM
* Correspondence: [email protected]
1
University of Liège, GIGA-Cancer Research, Human Genetics Unit, Liège,
Belgium
2
Division of Medical Oncology, Liège University and CHU Sart Tilman Liège,
Liège, Belgium
Full list of author information is available at the end of the article
© 2015 Boukerroucha et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Boukerroucha et al. BMC Cancer (2015) 15:181
DOI 10.1186/s12885-015-1205-1

occurring after mammary carcinoma, but their genomic
status concerning BRCA1 was unknown [10].
Here, we report two cases of patients with BRCA1 germ-
line mutation treated for breast cancer who developed glio-
blastoma few years after breast cancer diagnosis. The first
patient had a triple negative breast cancer (TNBC) and six
years later, a glioblastoma multiforme. In a very similar
pattern, the second patient developed also a triple nega-
tive breast cancer and five years later a glioblastoma. In an
attempt to clarify the role played by a mutated BRCA1
allele in the GBM development, we assessed BRCA1
mRNA and protein expression in the two tumour types
for each patient. We also checked the BRCA1 promoter
methylation status.
Ethical approval was obtained from the local institu-
tional ethical board (Comité d’éthique hospitalo-facultaire
universitaire de Liège) in compliance with the Helsinki
declaration, with the approval file number n°2010/229.
Cases presentation
Patient 1
A 28-years-old woman was diagnosed in 2000 with a
ductal carcinoma of the left breast.
After radical mastectomy, histologic analysis revealed
a 20 mm tumour with an infiltrating ductal carcinoma.
Immunologic analysis demonstrated no expression of
oestrogen and progesterone receptors (ER- and PR-) and
no overexpression of HER2.
The patient was staged as T1N0M0 stage IA and re-
ceived FEC adjuvant chemotherapy (FEC: Fluorouracil,
Epirubicin, Cyclophosphamide). The proliferation marker
Ki67 was expressed by 50% of the tumour cells.
Six years later, the patient developed a glioblastoma.
After complete macroscopic surgical resection, the tumour
was characterized as stage IV according to the WHO clas-
sification. The proliferative marker Ki67 was expressed by
40% of the tumour cells. The patient received temozolo-
mide chemotherapy and radiotherapy followed by chemo-
therapy alone (6 cycles).
Two years after her diagnosis of GBM, she developed a
carcinoma in the right breast. After mastectomy, the histo-
logic and immunologic analysis of the 26 mm tumour re-
vealed an infiltrating ductal carcinoma, negative for
oestrogen and progesterone receptors but with HER2 gene
amplification. The patient was staged as T2N0M0 stage
IIA and received adjuvant chemotherapy targeted therapy
(docetaxel and trastuzumab) for one year.
She died in 2012 after two relapses of the GBM.
BRCA1 genetic testing was performed after the first
breast cancer. The family tree is represented in Figure 1.A.
Patient 2
A56–years-old woman was diagnosed in 2005 with a
ductal carcinoma in the left breast. After mastectomy,
histological analysis revealed a 20 mm tumour with
an infiltrating ductal carcinoma. Immunologic analysis
demonstrated absence of hormone receptors expression
(ER-, PR-) as well as absence of HER2 overexpression.
Lymph nodes were not infiltrated. The tumour was clas-
sified as T2N0Mx stage IIA. The patient was treated
with 6 cures of FEC chemotherapy. She developed me-
tastases at the level of cervical and dorsal vertebra and
received palliative chemotherapy (paclitaxel) and zole-
dronic acid.
Considering the family history of the patient (Figure 1B)
and after molecular analysis of BRCA1 gene, the patient
was subjected to oophorectomy and hysterectomy.
Five years later, the patient developed a second breast
carcinoma in the right breast. After mastectomy, histo-
logic analysis revealed an 40 mm in situ ductal carcinoma
associated with a 2 mm infiltrating ductal carcinoma and
the absence of sentinel lymph node infiltration. Immuno-
logic analysis of the invasive carcinoma demonstrated the
absence of hormone receptors expression and an absence
of HER2 protein overexpression. The invasive tumour was
classified as pT1aN0Mx with a proliferative index based
on Ki67 expression of 35%.
Few days after tumour resection, the patient presented
mental confusion and a brain scan showed a mass at the
fronto-insular level. After surgical excision, the histologic
analysis revealed a glioblastoma characterized as stage IV
according to the WHO classification. The patient received
temozolomide chemotherapy and radiotherapy. One year
later, the patient had surgical resection of progressive
glioblastoma.
She died in 2011.
Molecular analysis
DNA isolation
Tumour determined by a pathologist was manually
macro-dissected from FFPE tissues. DNA was isolated
from the first triple negative breast carcinoma and
from the GBM in the two patients.
Blood samples were also collected in both patients to
establish the BRCA1 genomic status, after genetic coun-
selling. Genomic DNA from leucocytes was extracted by
standard phenol procedure.
BRCA1 gene analysis
All coding exons of BRCA1 gene were subjected to PCR
amplification. Amplicons were denatured, heterodu-
plexed and evaluated for the presence of mutations by
Denaturing High Performance Liquid Chromatography
(DHPLC) using product-specific melting and solvent
conditions. All amplicons showing abnormal DHPLC
pattern were sequenced by Sanger sequencing using
ABI 3130 and following manufacturer recommendations.
Boukerroucha et al. BMC Cancer (2015) 15:181 Page 2 of 7

The reference sequence BRCA1 (NM_007294.3) has been
used for HGVS-approved amino acid numbering.
The following pathogenic mutations were founded by
this method:
Patient 1: c.3481_3491del (p.Glu1161Phefs*3). This dele-
tion of 11 bp leads to a frame-shift and premature Stop
codon. This mutation has been previously identified in
our population.
Patient 2: c.2722G > T (p.Glu908*). This mutation in-
duces a premature Stop codon and is frequently identified
in our Belgian population (11% of our BRCA1 mutations).
These mutations were confirmed on independent sam-
ples by direct Sanger sequencing.
Moreover, the screening for large genomic rearrange-
ment was performed using Multiplex ligation dependent
probe amplification MLPA kits P002-C2 (MRC Holland)
on the BRCA1 gene region. This experiment did not
highlight any abnormal genomic copy number change.
In attempt to identify LOH, screening for the BRCA1
mutation has been performed by Sanger sequencing on
DNA extracted from cerebral tumours of the two pa-
tients. However, the DNA quality obtained from GBM of
patient 1 did not allow the amplification of the target
region and the subsequent BRCA1 sequencing. Regarding
patient 2, the BRCA1 mutation was identified in heterozy-
gous status, suggesting that no loss of heterozygosity
occurred.
BRCA1 and O
6
-methylguanine-DNA methyltransferase
(MGMT) methylation status
The methylation status of BRCA1 and MGMT promoter
was assessed by methylation specific polymerase chain
reaction (MSP-PCR) as previously described [11,12].
DNA extracted from tumours was first treated by so-
dium bisulfite using the EZ DNA Methylation Kit (Zymo
Research) following manufacturer recommendations.
MSP-PCR was done following the protocol of Esteller
and co-authors [13].
We assessed the methylation status of BRCA1 promoter
in the TNBC and GBM tumours for the two patients. A
methylation was observed in TNBC tumour but not in
GBM tumour for patient 1 as shown in Figure 2A. Regard-
ing patient 2, the BRCA1 promoter was unmethylated in
both tumours (Figure 2B).
On the other hand, the MGMT promoter was methylated
in GBM of both patients (Data not shown).
Figure 1 Family trees of the patients. A. Patient 1(arrow) B. Patient 2 (arrow). Cancer affected individuals are indicated.
Boukerroucha et al. BMC Cancer (2015) 15:181 Page 3 of 7

BRCA1 mRNA expression
The mRNA expression was assessed by in situ hybridization
using RNA scope technology (ACD) for FFPE samples.
In this experiment, the target probes are designed as
double-Z as described by Fay Wang and co-authors
[14]. The BRCA1 probe is complementary to the coding
region aa369-1482, spanning exon 5 to exon 11. To
quantify the expression, spots and cells were independ-
ently and blindly counted twice, in subzones of the
tumour. The ratio between the number of spots and
the number of cells was compared to those of a positive
control (MCF-7 cell line).
The mRNA expression level was 17,7% in breast car-
cinoma and 8% in GBM of patient 1.
Regarding patient 2, the mRNA expression level was
10,24% in breast tumour and 12,67% in GBM tumour.
Representative images of the BRCA1 mRNA expression
in breast tumour and in GBM of both patients are repre-
sented in Figure 3.
BRCA1 protein expression
The protein level expression was assessed by proximity
ligation assay (Duolink in situ detection reagents –
Sigma). This assay is more specific than the conventional
immunohistochemistry thanks to the use of two primary
antibodies directed against two epitopes of the same
protein. The primary antibodies are raised in different
species and are recognized by two secondary antibodies
coupled with oligonucleotide probes. After ligation of
the two probes, the circular DNA is amplified by poly-
merase reaction. The detection is performed using horse
radish peroxidase (HRP) labelled probes and a chromo-
genic reaction using 3,3’Diaminobenzidine (DAB). In
order to generate a signal only with the full length
BRCA1 protein, the two primary antibodies where
chosen to be specific to the N-term ([MS110] ab16780,
Abcam) and C-term domain (Sigma, SAB4502848) of
the BRCA1 protein, respectively.
The protein expression level was estimated using the
same quantification method as for mRNA expression
level, as compared to MCF7 cells.
The protein expression level was 4,08% in breast tumour
and 45,86 % in GBM of patient 1 and 19,04 % in breast
tumour and 36,37% in GBM of patient 2. Representative
images of the BRCA1 protein expression in GBM of both
patients are represented in Figure 4.
Promoter methylation, mRNA expression and protein
expression levels of BRCA1 are summarized in Table 1.
Discussion
Here, we report the cases of two patients, with a germline
BRCA1 mutation, who developed two primary breast can-
cers and a GBM. Both women present a germline patho-
genic heterozygote mutation in the exon 11 of the BRCA1
gene, leading to a truncated transcript.
In 80% of BRCA1 breast carcinoma the protein expres-
sion is lost because of the deletion of the second allele
[15,16]. Few cases of glioblastoma in BRCA1 mutation
carriers were reported but the BRCA1 expression status
has never been studied.
In an attempt to clarify the role played by a BRCA1
mutation in GBM development, we performed diverse
molecular experiments to characterize the expression
status of BRCA1 in glioblastoma, and in the first TNBC
of the two patients.
Our data show that the BRCA1 protein expression is
maintained in glioblastoma suggesting that no loss of
heterozygosity occurred in these tumours. The sequen-
cing data of the tumoural BRCA1 gene in the GBM of
patient 2 support this hypothesis. The protein expression
is tenfold higher in the glioblastoma of patient 1 than in
her breast carcinoma, and twice higher in patient 2.
However, the BRCA1 expression level was never com-
pletely lost, even in the TNBC. This observation is in
concordance with what is observed in breast cancer cell
lines where BRCA1 is mutated (HCC1937 ) or its pro-
moter methylated (UACC3199) [17,18].
BRCA1 expression is also known to be lost in some
sporadic breast cancers after methylation of the gene
promoter (13% of cases) [12,19], but promoter methyla-
tion is rarely observed in tumours of BRCA1 mutation
carriers [20]. However, we observed BRCA1 promoter
methylation in the triple negative breast carcinoma of
patient 1, but not in glioblastoma. In agreement with the
high protein expression level in the GBM, patient 2 did
not present any BRCA1 promoter methylation in this
Figure 2 Methylation status of BRCA1 promoter in tumours. A Patient 1. BPatient 2. M = methylated, UM = unmethylated, BC = breast cancer
tissue, GBM = glioblastoma tissue, MCF7 = MCF-7 breast cancer cell line known to have unmethylated BRCA1 promoter.
Boukerroucha et al. BMC Cancer (2015) 15:181 Page 4 of 7

tumour. Therefore, in these two cases, despite of a
BRCA1 pathogenic germline mutation, the tumour-
suppressor protein expression is maintained in GBM,
suggesting that the BRCA1 mutation is not instrumental
for GBM development. This observation is consistent
with cancer statistics that have not highlighted any in-
creased risk for brain tumour development in BRCA1
carriers [21,22]. However, these studies were conducted
in 1994 and 2002. Since then, treatment of breast cancer
has evolved and survival increased, maybe allowing the
detection of a previously hidden link between mutation
of BRCA1 and the risk to develop GBM. Indeed, as
breast cancer appears before GBM in BRCA1 mutated
patients, increased survival because of improved treat-
ments may now allow the recording of brain tumour
development. Moreover, the work of Konishi and co-
authors has demonstrated that a heterozygous mutation
of BRCA1 without loss of the wild type allele can still in-
duce genome instability [23]. Thus, conserved BRCA1
protein expression in glioblastoma does not completely
rule out its role in GBM development.
Another way to establish a relation between the BRCA1
mutation and the GBM development would be to estimate
the difference between the observed incidence of cases and
the theoretical occurrence risk of both events. In our Euro-
pean population and during the last 10 years of BRCA1
mutation screening, approximately between 2 and 8 cases
associating BRCA1 mutation and GBM for 100 million
women are expected [24-26], but only three cases have
been reported in the literature (two in Europe in the same
Figure 3 BRCA1 mRNA expression by in situ hybridization. A. Patient 1. Left : breast tumour; Right : GBM. B. Patient 2. Left : breast tumour;
Right : GBM. C. Positive control : MCF7 cells.
Boukerroucha et al. BMC Cancer (2015) 15:181 Page 5 of 7